1. Field of the Invention
The present invention relates to a method for controlling the carbon source concentration in aerobic cultivation of a microorganism. In particular, the present invention relates to a method for controlling the substrate carbon source that remains at a low level in a cultivation vessel during the culture feeding in aerobic fed-batch, continuous or cell-recycling continuous cultures. This is accomplished by monitoring the increase in pH or dissolved oxygen content in the culture media and adding the feed solution intermittently into the cultivation vessels at a calculated feed rate using a feed control device controlled by computer. Apparatus suited for practicing such a method is also provided.
The present invention further relates to a process for producing L-lysine by fermentation, which is an important amino acid used as a feed additive for broilers or pigs since L-lysine is in short supply in cereals for feed.
2. Background Information
For purposes of producing various substances by fermentation, for example, various amino acids or nucleic acids, using microorganisms or production of microbial cells for example, yeast cells, microorganisms are aerobically cultured. Aerobic cultivation of a microorganism is industrially performed by fed-batch culture, continuous culture or cell-recycling continuous culture, using carbon source(s) such as sugar, for example, as the main raw material.
In such a cultivation method, it is necessary to keep the concentration of carbon source (substrates) such as sugars in a cultivation vessel at a level as low as possible during the feeding of the culture. In this way several purposes are accomplished such as preventing substrate inhibition by the raw carbon source, reducing the loss of the raw carbon source by effectively utilizing the raw materials for the cultivation, easily isolating the product from the final fermentation broth, and preventing environmental pollution of the remaining carbon source contained in the waste liquid that remains after the product is isolated.
In the case of continuous culturing and cell-recycling continuous culturing, the product is isolated from the culture broth that is continuously discharged even during the feeding of cultures. It is necessary to control the flow amount of the carbon sources, for example, sugars, in the isolation process to the lowest level possible to make the influence of the carbon sources on the isolation process substantially zero. It is also necessary to prevent loss of the raw materials. In this type of cultivation, it has also been desirable to remove the manual operations for analyzing the carbon source concentration by substituting automatic monitoring that controls the stability of the carbon source concentration.
For maintaining the concentration of the carbon source, for example, sugar, at low levels during the feeding of the cultures, previously methods have been described which comprise controlling the carbon source concentration using an independent index such as the amount of oxygen consumed, amount of carbon dioxide gas discharged, pH, amount of the by-product produced, or amount of ammonia added, while adding carbon sources such as sugar in an amount obtained by multiplying them with a predetermined proportional coefficient. According to these methods, the microbial activity during the cultivation cannot be measured with high accuracy so that in some instances the concentration cannot be controlled satisfactorily when the activity has abnormally changed. For these reasons, it is impossible to effectively control the carbon source concentration at a low level (e.g., below 3 g/l) during the cultivation.
Furthermore, there is another known method which discloses detecting the exhaustion of carbon source concentrations in a cultivation vessel during cultivation only by the concentration of dissolved oxygen. However, sensing reliability is poor. Where the state of aeration or agitation (r.p.m. of agitation, an amount of air flow) changes, the concentration of dissolved oxygen greatly changes. In this case, the sensor sometimes incorrectly detects the exhaustion of carbon sources, whereby the carbon source concentration in the cultivation vessel cannot be effectively controlled. For these reasons, this method is not practical either.
Thus, it is clear that a need exists for a method and apparatus that automatically controls the carbon source concentration in the aerobic culturing of microorganisms. The present invention provides such a method and apparatus to overcome the aforementioned problems.
Another aspect of the present invention relates to a process for producing L-lysine by fermentation. Previously, methods have been described comprising the culturing of microorganisms capable of producing L-lysine by a batch or continuous process, accumulating L-lysine in the medium, and collecting the product, L-lysine.
In the case of batch processing, liquid medium containing carbon sources and nitrogen sources are put in a fermenter to perform incubation batchwise. Alternatively, medium containing the carbon source(s) alone are added continuously or intermittently to perform feeding of cultures.
In the case of continuous processing, incubation is carried out by supplying medium to a fermenter continuously and withdrawing the same volume of culture broth continuously to maintain the cell amount or concentration of the product, for example, at a constant level.
Where fermentation of L-lysine is performed by the conventional batch process, the accumulation of the product in the culture broth or the yields are high but there is difficulty in obtaining high productivity. On the other hand, when the conventional continuous process is used, the productivity is high but it is difficult to achieve high accumulation of product or high yields. In order to respond to an increased demand for L-lysine and prepare it at lower costs, it is necessary to enhance the productivity of L-lysine by fermentation and improve the concentration and yield of the product accumulated.
Thus, the need exists to provide a process for producing L-lysine by a fermentation method that alleviates the prior art's technical problems. The present invention combines the advantages of conventional batch processing and continuous processing by providing a novel fermentation method for producing L-lysine with high productivity, high concentration of accumulated product and high yield.